Extracellular secretion and targeted delivery by the type II secretion (T2S) system is considered a major virulence mechanism in gram negative pathogens, as many of the proteins secreted via the T2S pathway constitute important virulence factors, including toxins and degradative enzymes. The T2S apparatus is comprised of at least 13 different proteins, EpsC-EpsN and PilD, that assemble into a complex that spans the entire cell envelope of Vibrio cholerae. The dynamic and perhaps transient nature of this complex may be a prerequisite for function as its assembly and disassembly may drive extracellular secretion. The energy required for this process is thought to be generated from ATP hydrolysis by EpsE, a cytoplasmic protein that is associated with the cytoplasmic membrane via interaction with the membrane proteins EpsL. EpsM and EpsF. EpsE's interactions with these components modulate its ATPase activity and promote its localization to distinct sites within the V. cholerae cell envelope. The experiments described in this proposal are designed to test the hypothesis that specific protein-protein interactions and acidic phospholipids drive T2S in an ATP-dependent process at discrete sites In the cell envelope of V. cho/erae. Specffically, this proposal will i) determine the mechanism by which the enzymatic activity of EpsE is controlled by components of the cytoplasmic membrane including phospholipids. EpsL and EpsF;ii) investigate the ordered assembly of Eps components and determine the mechanism by which EpsD and EpsC drive focal assembly of the T2S complex;iii) map the cleft that forms when EpsM assembles and identify the cellular factor that binds to the cleft. Resolving the mechanisms of regulated assembly and spatial localization of the T2S system will further our understanding of T2S and may identify ways to manipulate the secretion process for preventative, therapeutic and/or biotechnological use.